This is a competing renewal application whose overall objective is to define the mechanisms of generation of the antileukemic effects of arsenic trioxide (As2O3) and to develop means and ways to enhance its antitumor properties. As2O3 is a heavy metal derivative with potent antileukemic activities in vitro and in vivo and it is highly effective in the treatment of patients with acute promyelocytic leukemia (APL). Despite advances in the field and the high interest to expand the use of arsenic to other hematological malignancies, the precise mechanisms by which As2O3 induces antileukemic responses are not known. We have provided the first evidence that As2O3 -induced autophagy is essential for generation of its suppressive effects on primitive leukemic progenitors from patients with myeloid leukemias. The current proposal is a systematic approach to define the cellular events that lead to As2O3 -dependent autophagy and to identify the mechanisms of autophagy-mediated degradation of oncogenic proteins in primitive leukemic precursors and leukemia initiating stem cells (LICs). Specific aim 1 will examine the mechanisms of As2O3 -autophagy in leukemic progenitor cells. Studies will be performed to identify upstream kinases in the autophagic cascade in leukemia cells and to define the sequence of events leading to their activation in response to arsenic. Other studies will examine the relationship of autophagy with cellular negative feedback regulatory pathways (NFRPs) that are induced in response to As2O3. Specific aim 2 will identify effector mechanisms by which autophagy mediates arsenic- responses on primitive progenitors and leukemia initiating stem cells from patients with AML and CML. Experiments will be performed to define whether autophagy mediates arsenic-dependent degradation of different oncoproteins and to define the precise mechanisms by which this occurs. Other studies will determine whether pharmacological or molecular targeting of elements of the autophagic machinery can enhance arsenic-induced suppression of primitive leukemic precursors and LICs. Specific aim 3 will examine the role of autophagy in arsenic-dependent antileukemic responses in different leukemia mouse models in vivo. It involves experiments to determine whether autophagy is required for the antileukemic properties of As2O3 in vivo and to examine whether other inducers of autophagy exhibit synergistic antileukemic activities with As2O3. Altogether, this work will advance our understanding of the mechanisms by which As2O3 generates antileukemic responses. It should have important clinical-translational implications and possibly lead to the development of novel approaches to target early progenitors and LICs using combinations of As2O3 with other autophagy inducers.